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Book your room at La Quinta Inn & Suites Durham Research Triangle Park
Get an overview of this hotelHotel in Durham with free breakfast and outdoor pool
Situated in Durham, this hotel is 2.6 mi (4.3 km) from Herndon Hills Farm and within 6 mi (10 km) of Streets at Southpoint Mall and North Carolina Central University. Lakeshore Golf Course and Forest Hills Park are also within 6 mi (10 km).
Hotel Features
An outdoor pool, a fitness center, and a spa tub are available at this hotel. Free continental breakfast, free WiFi in public areas, and free self parking are also provided. Additionally, coffee/tea in a common area, a seasonal outdoor pool, and a business center are onsite.
Room Amenities
All 141 rooms offer free WiFi, premium bedding, and flat-screen TVs with cable channels. Guests will also find coffee makers, free local calls, and hair dryers.Would you like to give us feedback? Share it! Opens in a new window
La Quinta Inn & Suites Durham Research Triangle Park1910 Westpark Dr Durham NC 27713
Hotel Amenities
Hotel Amenities
In addition to an outdoor pool, La Quinta Inn & Suites Durham Research Triangle Park provides a spa tub and a fitness center. High-speed wireless Internet access is complimentary. Business-related amenities consist of a business center and a meeting room. A complimentary breakfast is offered. Event facilities measuring 500 square feet (46 square meters) include conference space. This Durham hotel also offers barbecue grills, multilingual staff, and a garden. Complimentary self parking is available on site.
- 24-hour front desk
- Barbecue grill(s)
- Business center
- Coffee/tea in common areas
- Conference space
- Conference space size (feet) - 500
- Conference space size (meters) - 46
- Dry cleaning/laundry service
- Elevator/lift
- Express check-out
- Fitness facilities
- Free breakfast
- Free self parking
- Free WiFi
- Garden
- Multilingual staff
- Number of floors - 6
- One meeting room
- Outdoor pool
- Outdoor seasonal pool
- Safe-deposit box at front desk
- Spa tub
- Television in common areas
- Total number of rooms - 141
Family Friendly Amenities
- Cribs/infant beds (complimentary)
- Free Wi-Fi
- Outdoor pool
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Air conditioning
- Cable TV service
- Coffee/tea maker
- Free toiletries
- Free cribs/infant beds
- Daily housekeeping
- Desk
- Flat-panel TV
- Free local calls
- Free WiFi
- Hair dryer
- Iron/ironing board
- Premium bedding
- Premium TV channels
- Private bathroom
- Shower/tub combination
Where to Eat
Guests are offered a complimentary continental breakfast.
Nearby Things to Do
An outdoor pool and a spa tub are on site. Other recreational amenities include a fitness center.
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- Accessible bathroom
Hotel Policies
Check-in time starts at 3 PM
Check-out time is Noon
Payment types
- Pets stay free
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No rollaway/extra beds available
Pets allowed (no fees)
Hotel Name
- Quinta Durham Research Triangle Park
- Quinta Inn Durham Research Triangle Park
- Quinta Inn Triangle
- Quinta Triangle
- Quinta Inn Triangle Park
- Quinta Triangle Park
We should mention
Children 18 years old and younger stay free when occupying the parent or guardian's room, using existing bedding.


text:
Bob's Daily Buzzword: `Sterilization'
Sept. 5 (Bloomberg) -- Bob Rice, general managing partner with Tangent Capital Partners LLC, explains the "Sterilization." (Source: Bloomberg)
Most Recent Videos
Britain Needs Keynesian Stimulus, Says Dryden
13:24 - As Bank of England officials gather this week to decipher the latest post-Brexit reports, they'll probably assess a nasty outlook of slowing growth and faster inflation. Varying levels of tolerance for accelerating price growth are likely to be a major theme of the debate and are already dividing the nine-member Monetary Policy Committee on the need for stimulus. JPMorgan Asset Management Global Market Strategist Alexander discusses with Bloomberg's Anna Edwards and Manus Cranny on "Countdown."

subset: commoncrawl


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After you install this plug-in (it only takes a minute or so), each time you start InDesign you'll see a new tip or technique appear on your screen. Enjoy! The CS5 update for the Free version will not be released. If you enjoyed your daily tips in InDesign CS - CS4 and want to keep getting them in CS5, you need to get the Blatner Tools plug-ins suite.
Imposer Pro - plug-in for Adobe InDesign that imposes document pages into printer flats. Imposer Pro offers six sheet types (2-Up, 4-Up, 8-Up, 2-Up Consecutive, 3-Up Consecutive, and 4-Up Consecutive), four binding types (Saddle Stitch, Perfect Bound, Stacked, and None) and five imposition types (Standard, Work and Turn, Work and Tumble, Split Web, and Sheet Wise). To meet all your imposition needs — such as using different imposition methods for the same document — you can choose which pages to impose and how many signatures to create.
MathMagic Pro Edition for Adobe InDesign is an equation editor mainly for use with Adobe InDesign software in editing any mathematical expressions and symbols with WYSIWYG interface and various powerful features. The equations can be verified and tested.
If you are looking for an application to convert your image files into the popular PDF format, then this software is undoubtedly for you. This program allows you to convert image files such as JPG, TIF, PNG, BMP, WMF, DIB and EMF directly to PDF file. Thanks to this program, you will be able to create interesting PDF albums with the page transition effects.
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This software offers essential tools for producing everything from professional page layouts to rich interactive experiences in a unified environment that lets you work more productively than ever before. Create web and interactive content and design websites. Enjoy new creative effects and controls, as well as productivity enhancements such as Multi-file Place and table and cell styles.
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FotoMorph is a small yet powerful utility that can transform your static photographs into dynamic and animated Flash videos while adding stunning morphing, warping, and panning effects to them. To top it all off, you can also add transitions and loops that you can finally export as AVI or SWF videos, a GIF animation, or an HTML page with a Flash video embedded in it.
Evrsoft First Page 2006 3.0 is a free HTML web page editor.
It comes bundled with some templates, as Basic HTML Document, CSS (Cascade Style Sheets), HTML 4.01 and 4.01 Transitional and XHTML Strict, Mobile and Transitional. It also has design elements, like scroll bars, forms, FAQs and glossaries, as well as Dynamic Pages Templates (Java, JSP, PHP and XML) and Legal Forms.
2D&3D Animator produces high-quality images for your Web page. It enables you to create animations from your own pictures and add 3D texts and shapes. Output may be exported as GIF, AVI, JPEG, ICO, PNG or BMP clips and images.
Visual Slideshow is a simple and neat tool that offers a fast and easy way of creating photo slideshows and publishing them on the web.
One of its most remarkable advantages is the simple, intuitive and straightforward interface. It manages to be self-explanatory and easy-to-use while still looking quite stylish.
AnvSoft's Photo Slideshow Maker Professional produces high-quality animated slideshows out of your static photo albums. The resulting Flash-based presentation can be then burn to CD or DVD, distributed via e-mail to your friends and family, shared on your personal website, or uploaded to AnvSoft's own cloud service, Go2Album.
With BeCyPDFMetaEdit, you can edit any of the metadata embedded in a PDF document. Besides, you can add information that is not already available in the original file, such as new keywords, bookmarks, or page labels. You can also customize the transition between pages when using the PDF document as a slide show.


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WASHINGTON, DC—Expect the talk at this year's gathering of financial luminaries in Jackson Hole, WY to be all about interest rates and not just because its theme for 2014 is "Re-evaluating Labor Market Dynamics."
Simply put, the direction of interest rates—and when will they finally start to rise—is among the most important economic issues for the year. Interest rates, of course, could be called a perennial issue of importance, but 2014 is a shade difference as a contingent of economists, businesspeople and policymakers become more worried that the Federal Reserve Bank is waiting too long to pull the trigger.
For instance, private economists surveyed by the Wall Street Journal overwhelmingly said they are more concerned the Federal Reserve will hold off on raising rates too long than they are of the Central Bank raising them too soon or too quickly.
At the same time, though, there is another cloud looming on the horizon, one that is coming in perhaps faster than the specter of rising rates. Construction costs have risen sharply in recent months with the price of steel alone jumping some 40% over a 90-day period. Consulting firm Rider Levett Bucknall says US construction costs rose 1.15% in the first quarter of 2014, the largest three-month increase since 2008.
Our question, on Jackson Hole eve, is which is worse for the industry: the uncertainty around interest rates or these rising building costs.
Another question, as suggested above, is what do people think will happen with interest rates, both this week and the coming year.
The argument for the Fed to start raising rates sooner rather than later is straightforward: the recession is long over and the unemployment rate has from 7.3% last year to 6.2% today.
The argument for the Fed holding off on raising interest rates is equally compelling: there is little indication of inflation in the economy and more importantly, wages are not rising and jobs that are being created after the recession do not pay as much as the ones lost. In addition, yields on US Treasurys are at a fourteen-month low thanks to investors seeking a safe haven from global turmoil—but also signaling a confidence in the Central Bank.
Please come back here throughout the week as we will be examining all of these questions – about rates, about how badly will construction costs hurt development—and a related question about whether rising rents can mitigate the aforementioned.
Stay tuned for part 2


text: Harper, Ford talk gun crime Add to ... The Globe and Mail Published Tuesday, Jul. 24, 2012 6:15PM EDT Last updated Tuesday, Jul. 24, 2012 6:17PM EDT Prime Minister Harper says he and Toronto Mayor Rob Ford discussed tough penalties for gun crime at a meeting Monday, but he wouldn't go into much detail. Ford requested the meeting after a recent spate of shootings in the city Watch More Video: Trump: Clinton should have congratulated me for making history Funeral for Ottawa man who died after police altercation He-said-she-said: Contrasting Trump and Clinton's convention speeches Bad boy 'Justin' beaver terrorizes B.C. town Two San Diego police shot, one killed 'No Donald, you don't': Watch the highlights from Hillary Clinton's convention speech Chelsea Clinton on her mother - 'my role model' - and Triceratops Hillary Clinton accepts the Democratic nomination with 'humility, determination' 'I will gladly lend you my copy': Father of fallen Muslim soldier offers Trump his Constitution Clinton has 'got a friend' in Carole King Fixing payroll debacle will cost up to $20 million: Official 'He destroyed our family': Sammy Yatim's mother says her anger hasn't dissipated Const. Forcillo sentenced to six years in shooting death of Sammy Yatim Sarandon, Glover, Woodley protest DNC snub Three Kentucky inmates walk out open jail door; one still missing Pope Francis tumbles at World Youth Day ceremony Video: Obama asks America to 'reject cynicism and reject fear' Biden says Trump's stated concern for middle class is 'bunch of malarkey' 'Russia, if you're listening': Trump calls on hackers to find Clinton's missing emails Trump denies involvement in email hack, impersonates Jon Lovitz Comments AA More Related to this Story Canada's crime rate at lowest level since 1972 Harper to meet with Toronto's Mayor Ford to discuss crime Ontario pledges $12.5-million to combat gun violence, but won't pay for more police Follow us on Twitter: @globeandmail


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Regulation of Arf Activation: the Sec7 Family of Guanine Nucleotide Exchange Factors
Version of Record online: 7 AUG 2007
DOI: 10.1111/j.1600-0854.2007.00634.x
Volume 8, Issue 11, pages 1476–1485, November 2007
Additional Information
How to Cite
Casanova, J. E. (2007), Regulation of Arf Activation: the Sec7 Family of Guanine Nucleotide Exchange Factors. Traffic, 8: 1476–1485. doi: 10.1111/j.1600-0854.2007.00634.x
Publication History
- Issue online: 7 AUG 2007
- Version of Record online: 7 AUG 2007
- Received 15 June 2007, revised and accepted for publication 3 August 2007, uncorrected manuscript published online 7 August 2007, published online 10 September 2007
- Abstract
- Article
- References
- Cited By
- Arf;
- nucleotide exchange;
- Sec7
The ADP ribosylation factors (Arfs) are a family of small, ubiquitously expressed and evolutionarily conserved guanosine triphosphatases that are key regulators of vesicular transport in eukaryotic cells (D'Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 2006;7:347–358). Although Arfs are best known for their role in the nucleation of coat protein assembly at a variety of intracellular locations, it is increasingly apparent that they are also integral components in a number of important signaling pathways that are regulated by extracellular cues. The activation of Arfs is catalyzed by a family of guanine nucleotide exchange factors (GEFs), referred to as the Sec7 family, based on homology of their catalytic domains to the yeast Arf GEF, sec7p. While there are only six mammalian Arfs, the human genome encodes 15 Sec7 family members, which can be divided into five classes based on related domain organization. Some of this diversity arises from the tissue-specific expression of certain isoforms, but all mammalian cells appear to express at least six Arf GEFs, suggesting that Arf activation is under extensive regulatory control. Here we review recent progress in our understanding of the structure, localization and biology of the different classes of Arf GEFs.


text:
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text:
Hotels near Memorial Presbyterian Church
Search hotels near Memorial Presbyterian Church in St. Augustine
Last minute St. Augustine deals
- Magnuson Hotel Historic St. Augustinefrom $100$85
- St. Augustine Island Innfrom $70$63
- The Loft at Menorcan Squarefrom $269$135
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"Great location. Good pool. Good restaurant and great staff"
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32 people looked at this hotel in the last hour
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"Relaxing stay after a tropical depression passed through. Fla. in the late spring and summer there is always a chance. We had a shaky start with a plumbing problem with the room we had booked. Fortunately they had another room available. Once repaired we were moved and had a pleasant stay. Break..."
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19 people looked at this hotel in the last hour
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"I was welcomed early before noon and I was told I could park there. Even after I checked out the next morning they permitted me to park there while I participated in a boat trip. I would have had to walk a long way to get to the harbor or find parking by a meter."
A Traveller, May 2016, US
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"We had a great time in St Ausgustine. The staff were very friendly and accommodating to our needs. They provide a full hot breakfast each morning, late afternoon social hour with drinks and light snacks and evening dessert which can be waiting in your room if you're out for the evening. We were m..."
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"Jackie and Kelly were tremendous!!!!! I highly recommend 63 Orange to anyone. The rooms were comfortable and the hospitality is second to none!!!1 Loved it!"
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"We really appreciated the entire stay. We were looking forward to going to visit Old St. Augustine and not have to move the car . . . and we didn't. We parked on July 1 and didn't need to move it again until we left on July 4. The innkeepers were very helpful - even brought our luggage to our ups..."
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"Excellent stay! Staff was wonderful and accommodating. Room was well kept and comfortable. Complimentary coffee, wine & baked goods available in lobby. Great location in the middle of St. Augustine. Would stay again in a heartbeat!"
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"Location, Location. Friendly staff, very clean and a short walk to St. George Street. Parking is for small, small/medium cars in the back. Good luck parking on the street. We'll be back. Our experience made us agree we need to frequent St. Augustine more."
A Traveller, Aug 2015, US
- Agustin InnNo Guest Reviews yet
Map of hotels near Memorial Presbyterian Church
- Vilano Beach
- Old St. Augustine Village
- Castillo de San Marcos
- Anastasia State Park
- Flagler College
- World Golf Village
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- Marineland
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- Old City Gates
- Fort Matanzas National Monument
- Fountain of Youth Archaeological Park
- Cathedral Basilica of St. Augustine
- Lightner Museum
- St. Augustine Pirate and Treasure Museum
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text:
Steeped In Cash
Last Sunday, Rep. Ron Paul hosted an online "tea party." His supporters dumped nearly $6 million into the congressman's campaign coffers. (That is the largest sum a candidate for president has raised in a single day). Chris Cillizza, of washingtonpost.com, tried to figure out what Rep. Paul will do with all that money. And he wondered why the campaign can cultivate donations, but few new supporters. Bret Hayworth, of the Globe Gazette, in Mason City, Iowa, asked the same question.
In the first hour, our "Political Junkie," Ken Rudin, joins us again, as he does every Wednesday, to talk about politics and the campaign for president. As usual, [almost] anything goes! Leave us your questions. Send us your comments. We're going to talk about Rep. Ron Paul, then we'll turn our attention to Iowa. David Yepsen, of The Des Moines Register, will join us. If you live in "the Hawkeye State," are you excited about the caucus? (If you can't wait for the political season to be over with, tell us why).
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Please keep your community civil. All comments must follow the NPR.org Community rules and terms of use, and will be moderated prior to posting. NPR reserves the right to use the comments we receive, in whole or in part, and to use the commenter's name and location, in any medium. See also the Terms of Use, Privacy Policy and Community FAQ.
I really wanted to ask David Yepsen why his paper didn't invite Kucinich or Gravel to their democratic debate (supposedly because they don't have store-front campaign offices in Iowa) but Alan Keys (who does NOT have an office there, much less a real shot at nomination) took part in the republican debates the day before. Sadly all I got was the busy signal.
Funny but I think the support is there and the measuring method is broken.
I confused as to why these commentators don't see 24,000 new donors as 'new supporters' of the Ron Paul campaign. Am I missing something?
The question I'd like to ask is what Ken Rudin believes it would take for Ron Paul to win the Republican nomination.
I was very disappointed with the coverage of the one-day record set by Dr. Ron Paul's supporters. The show spent less than two minutes discussing the significance of the unprecedented outpouring of grassroots support for Dr. Paul. I expected much better coverage from NPR.
There is a real revolution taking place in this country. Dr. Paul supporters are citizens who are fed up with the status quo of both the Republicans and Democrats. These supporters are disgruntled with Washington, D.C and previously apathetic towards the political process. For example, the supporter who independently organized both of the record online fundraisers has never voted before. These supporters are not on the polling lists
nor being taken into account by political pundits.
Dr. Ron Paul in his own words:
On just one day, in honor of the 234th anniversary of the Boston Tea Party, the new American revolutionaries brought in $6.04 million, another one-day record. The average donation was $102; we had 58,407 individual contributors, of whom an astounding 24,915 were first-time donors. And it was an entirely voluntary, self-organized, decentralized, independent effort on the internet. Must be the "spammers" I keep hearing about!
The reasons Ron Paul hasn't done well in the polls are these: 1. He has been excluded from many of them. If you look, quite a few of them don't even have him as an option. 2. The Republican Party members who are polled are often only those with land lines. They usually do not call cell phones, and many of Paul's supporters are of the 'cell phone only' crowd.
Also, some online polls show him doing excellent, however those polls are often 'missing' soon after they are taken.
One CBS online poll was attached to an article talking about how well Huckabee was doing yet when a person takes the poll it showed Paul at 90% or better!
Where's the coverage of Ron Paul in this segment? You headline him online, but in the actual conversation you talk about him for less than 20 seconds. Shame on you NPR, for continuing to ignore the only GOP candidate worth voting for.
Steeped in something, But I'm sure its NOT cash.
Who would be his running mate, Joe Lieberman? This man is NOT a viable party nominee. (Not that I care, but) "The powers that be" within the party will not back him.
Prometheus to Pandora: Who cares about the animals NOW, at least we have HOPE.
From: AP
Democrats came to Washington promising to clean up the place, but the 9,000 earmarks, or pet projects, in the newly passed omnibus spending bill suggest that the mess wasn't just a Republican problem.
Where are the Republicans? Where is Ron Paul? They too are COMPLICIT!
Doctor Ron Paul is the real deal.
He has the cure for America!
Peace, Prosperity, Liberty. A true Classical Liberal dressed up as a Conservative. -the best of both worlds!
Thanks NPR for your coverage of this decade's 'phenomenon'! :)
NPR coverage of Ron Paul has been pathetic to say the least. I for one am no longer donating to NPR and have redirected my funds to Ron Paul's campaign.
I love you NPR, but your Ron Paul coverage needs to change. If it does, I may consider becoming a member again.
I agree that there is a real revolution in this country. The internet is helping build awareness of the wasteful and often malignant results of forced societal engineering by the U.S. government.
The dinosaurs in power should wake up and notice their eggs being eaten by the small, more intelligent mammals lest they become as extinct as the non-metaphorical dinosaurs.


text:
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My Long Jourrney to Lose Weight
Last year, my life was a mess. I lost a lot of money in a business investment and I became depressed. Nothing could cheer me up. I turned to food as a way to drown my sorrows. I did not care about taking care of my body. I ate some of the unhealthiest foods to ever exist. My favorites were fried chicken, chili-cheese fries, and barbecue ribs. I had these about every week. After a major health scare, I realized I had to take back control of my life. I purchased a Nutri Ninja to help me lose weight. I knew that I had to make a change.
While I was depressed, eating a healthy diet was the last thing on my mind. I had no desire to cook. (more…)


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Endurance exercise and metabolism are linked to transcriptional activation of human telomeres, researchers propose.
Animals as art
February 23, 2007|
A biologist and journalist review a Brooklyn artist's attempt to bring the zoo to SoHo
February 23, 2007|
A biologist and journalist review a Brooklyn artist's attempt to bring the zoo to SoHo
February 23, 2007|
More than half of the 13 field labs, which inspect food and drugs, could be shuttered
February 21, 2007|
A microbicide designed to ward off HIV is unexpectedly linked to an increased risk of infection, prompting speculation about the future of microbicides against HIV
February 20, 2007|
A 2005 paper about premature ovarian failure is identical to a paper published in a Korean journal
February 19, 2007|
In a study some experts say largely confirms previous work, a Japanese team shows new teeth can develop in the adult mouse mouth
February 16, 2007|
A recent PhD pens a wildly popular online novel based on Phantom of the Opera
February 16, 2007|
The age-old practice may harm both science and scientists
February 16, 2007|
The institution closes its health research facility and plans a $20 million renovation to maintain accreditation
February 15, 2007|
There's more evidence that neurogenesis occurs in the human olfactory bulb, but what do these new neurons actually do?
February 14, 2007|
A University of Missouri probe of a Science paper exonerates the lead author and two co-authors, but a fourth author is on the lam, and still under scrutiny


text:
Latest Headlines
The diverging video strategies of Verizon Communications and AT&T will be on display this week as the companies' finance chiefs face investors. On the one hand, AT&T will have a chance to flesh out its plans to integrate DirecTV following the close of its $48.5 billion deal in July for the pay-TV provider. Meanwhile, Verizon is likely to discuss the early returns of its Go90 over-the-top mobile video service, which launched nationwide to all wireless customers earlier this month.
Dish Network and Verizon Communications could strike a deal by year-end for Verizon to get access to Dish's spectrum through a leasing arrangement, according to analysts at New Street Research.
A number of New York City officials said they are considering suing Verizon for not meeting their proposed FiOS buildout obligations set in their 2008 franchise agreement.
Eric Schneiderman, New York's attorney general, has written a letter to the New York Public Service Commission (NYPSC) asking the state regulator to investigate Verizon and look at how the deregulation of the New York telecom market is impacting businesses and consumers.
Phone Recovery Systems filed a lawsuit in Massachusetts against Verizon Communications, Comcast and other phone companies claiming that the firms have failed to pay the state $214 million in fees to support the 911 system.
Verizon's troubles in New York City continue to mount as Mayor Bill de Blasio is requiring City Hall to approve any business local agencies do with the service provider, a measure focused on getting it to fulfill its goal to wire the city with FiOS FTTP service.
Verizon is coming under fire again in New York City over claims that the service provider is preventing competitors' broadband data service from being delivered to their customers by blocking access to necessary fiber conduits.
Verizon Communications has no interest in buying Dish Network, according to Verizon CFO Fran Shammo.
Cablevision is suing Verizon Communications in a Manhattan federal court claiming that its TV commercial that says Verizon's FiOS is not 100 percent fiber optic cable is truthful.
Verizon Communications' $4.4 billion acquisition of AOL, which a top Verizon executive has said is mostly about AOL's advertising technology, is raising new concerns that customers' privacy will be exploited in Verizon's push to create more targeted ads.
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text:
Robert L Castor Post 5101
Veterans of Foreign Wars
getting HOT out there!
So is our Post!
State Honors and 1st in District!
Post Officers:Commander - Bruce Levering
Sr Vice - James L. Mock
Jr Vice - Emory Estep
Quartermaster- Grover Roach
Adjutant-Emory Estep
Judge Advovate - Floyd W Johns
Service Officer-Bruce Levering
Chaplain- Heather E Capra
Surgeon - Jim Mock
1st Yr Trustee - Justin Teeter
2nd Yr Trustee - Floyd Johns
3rd Yr Trustee - Kurt Hensley
Check our Canteen Page
for Banquet Hall Information
Post Current NewsNew Members Needed!
Officer Elections!====================================================
Thank you to all the new officers who have stepped forward to help keep our post growing. Thanks to the efforts of the officers of the past year, we have received State Honors and First in District Honors. Let's keep the string going and welcome and help the new officers take us even further.
!Pastoral in the Park!
Independence Day Celebration in Lexington at Bicentennial Park: a family and patriotic picnic will be held from 10:30am till 2:30pm. We are a primary sponsor and will have a visible presence at the event, so come on down and join us for music, food, contests and more. You won't believe what we have done in four short months. Check here for more information.
New Members Joining!
We would like to welcome the new members joining our post. Please make them feel welcome when they stop by. We know you will!
Do we make a difference?====================================================
Read This.
We received this letter from the Principal of Central Elementary in Lexington, Ohio. A thank you for our support of their efforts
It's July now, only three months seperate us from Post breakfasts. Sorry crew, hurrah for fans. Fortunately for us that also means that Breakfasts are now available at the post. Come in Saturday and enjoy our famous breakfasts, Omelets, Pancakes, Eggs and Toast, or our Famous Mess. Want to know your order before you get here? OK, here's a menu.
Want to learn about our History?
Al Rinehart gave the post an album from his time, back when. When he was a young Navy man in WWII. Every one of us should check it out. This is the reason we exist. We are a place for veterans to call home. We are a place where veterans don't have to worry about their attitude on a night. We are a place where a veteran can find someone who can listen with a sympathetic ear. Even if that person hasn't been in the exact same situation, they can relate and begin the process, of allowing that veteran to heal, serve, celebrate.
Friday Night is Pizza & Wing Night
Ask Grover about the Kitchen Sink Pizza. New staff, same great taste! We are selling pizzas every Friday evening from 5pm till 7pm. Check out our latest wings menu or pizza menu!
We are also celebrating with free jukebox music and other specials. Check it out and ask.
Let Us Know How You Are!
Hey people! Stop by and say hello. If you haven't been here for a while, we'd like to see your face. Stop by on Friday for a pizza or on Thursday for cornhole. Just let us know you are still with us. We are working hard to make this post better than it has been in a decade. Come check us out and see where you can fit in. Maybe you can even make a difference by helping out somewhere???
Contact Us!Let us know how we are doing! We can be contacted at the post using this address.
Do you know anyone who is eligible to become a member of our post, in any capacity, Member, Auxiliary, or Social? All are needed for the ongoing health of the post. Please introduce them to us and give them and us a chance to keep a great post going.
On the Lighter Side
Have you heard of The Onion, America's Finest News Source? It is a wonderful source of Satirical News. I just recently discovered a military focused cousin to that site, The Duffle Blog, written by and for veterans and their families. If you need a break or just want a few laughs, check either one out.
Need a Flag?!?!?
Did you know that our post has US Flags for sale? We do! They are 3 Feet by 5 Feet, and cost $25.00. You can arrange to pick one up by calling the post at 419-884-2873.
Never Forget our Purpose. Please keep in mind our family serving overseas at the current time. Even if our family members and friends are home, there are MANY still serving in the line of fire for our freedom. Please remember them all.
Thanks for stopping by! If you have any pictures to share or pictures you would like posted of VFW Post events here, email them to email@example.com with a caption you would like posted! If you have an idea to help out, just let us know, fund raising, volunteering, sources of materials.... all suggestions are welcome.
© 2009, 2015 Robert L Castor Post 5101 Veterans of Foreign Wars. This site created by and currently maintained by James Dolan, Mens Auxiliary Chaplain with the supervision of members of the post and officers of the regular membership.


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Tea Towel Tuesday: Take 15% off 1+ yards of Linen Cotton Canvas Ultra Through 11:59 PM EDT July 26th.
I'm actually enjoing everything I got time to do with my finguers! sewing and illustrating are my favorite craft works, that's why spoonflower is such a perfect place for me!
© 2008-2015 Spoonflower Inc


text:
StartUpHire is the place to find DIRECTOR, CMC REGULATORY AFFAIRS jobs. Review the list of DIRECTOR, CMC REGULATORY AFFAIRS career opportunities to the left. These DIRECTOR, CMC REGULATORY AFFAIRS jobs represent some of the most rewarding careers available.
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Requisition Number: 16-918 Job Title: Vice President, Regulatory Affairs City: Rockville State: MD Vice President, Regulatory Affairs Apply Now Email Description/Job Summary MacroGenics is a leader in the discovery and development of innovative medicines that utilize our next6/2/2016 Rockville, MD Save This Job
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Exped DreamWalker DUO 400 Sleeping Bag - Double
- Lightweight down-insulated sleeping bag is used together with the included sheet and 2 sleeping pads (sold separately) to create a multifunctional double bed
- Cover 2 sleeping pads (sold separately) with the included sheet, then snap the DreamWalker to the sheet to keep it in place throughout the night
- Slide your feet inside the built-in foot bag at the bottom of the DreamWalker for extra insulation; if too warm, stretch your feet out under the foot bag
- Side baffles on the quilt prevent cold air from entering along the edges
- Soft sheet is comfortable next to skin; mesh sides prevent moisture on the floor of your tent from migrating up the sheet
- Drawcords keep the sheet snug around your sleeping pads
- Flip the DreamWalker in half and snap it to itself to create a rectangular down sleeping bag for solo use
- Wear the DreamWalker DUO as an insulated poncho to keep you warm while you're hanging around camp; zippered slit in the middle of the quilt serves as a head opening
- 2 zippered pockets allow each person to store small items within reach
- The Exped DreamWalker DUO 400 sleeping bag includes a stuff sack and storage sack
- Size M and Large Wide differ only in the size of the sheet; size M sheet fits over 2 Exped size M mats; size Large Wide sheet fits over 2 Exped Large Wide mats
- Pillows and sleeping pads that are pictured with the sleeping bag are sold separately
REI membership
|Best Use
|Temperature rating (F)
|45 degrees Fahrenheit
|Temperature rating (C)
|7 degrees Celsius
|2 lbs 10.3 oz. / 2 lbs 13.7 oz.
|Insulation type
|700-fill duck down
|Fill weight
|17.6 / 17.6 ounces
|Sleeping bag shape
|Fits up to (in.)
|72 / 77 inches
|Shoulder girth (in.)
|86 / 86 inches
|Hip girth (in.)
|86 / 86 inches
|Stuff sack size
|9.8 x 12 inches / 9.8 x 12.6 inches
|Stuff sack volume
|6 / 7 liter


text:
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Definition of to hell in English:
to hell
Used for emphasis: damn it to hell
More example sentences
- She hoped to hell that Thomas would ask her if she would help him.
- I have never seen him like that before, and I hope to hell that I never have to again.
- She just had to keep talking to him, and hope to hell that the ambulance would get here soon.
Definition of to hell in:
What do you find interesting about this word or phrase?
Comments that don't adhere to our Community Guidelines may be moderated or removed.
= trending


text:
Secure multiparty computation of approximations
Full Text:
Get this Article
Joan Feigenbaum
Yale University, New Haven, CT
Yuval Ishai
Technion, Haifa, Israel
Tal Malkin
Columbia University, New York, NY
Kobbi Nissim
Ben-Gurion University, Beer Sheva, Israel
Martin J. Strauss
University of Michigan, Ann Arbor, MI
Rebecca N. Wright
Stevens Institute of Technology, Hoboken, NJ
2006 Article
· Downloads (6 Weeks): 2
· Downloads (12 Months): 30
· Downloads (cumulative): 808
· Citation Count: 31
Published in:
· Journal
ACM Transactions on Algorithms (TALG)
TALG Homepage
Volume 2 Issue 3, July 2006
Pages 435-472
New York, NY
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Secure multiparty computation of approximations
Secure multi-party computation
Secure multi-party computation (also known as secure computation or multi-party computation) is a sub field of cryptography. The goal of methods for secure multi-party computation is to enable parties to jointly compute a function over their inputs, while at the same time keeping these inputs private. For example, two millionaires can compute which one is richer, but without revealing their net worth. In fact, this very example was initially suggested by Andrew C. Yao in a 1982 paper.
more from Wikipedia
An approximation is a representation of something that is not exact, but still close enough to be useful. Although approximation is most often applied to numbers, it is also frequently applied to such things as mathematical functions, shapes, and physical laws. Approximations may be used because incomplete information prevents use of exact representations. Many problems in physics are either too complex to solve analytically, or impossible to solve using the available analytical tools.
more from Wikipedia
Computing the permanent
In mathematics, the computation of the permanent of a matrix is a problem that is believed to be more complex than the computation of the determinant of a matrix despite the apparent similarity of the definitions. The permanent is defined similarly to the determinant, as a sum of products of sets of matrix entries that lie in distinct rows and columns. However, where the determinant assigns a ±1 sign to each of these products, the permanent does not.
more from Wikipedia
#P-complete, pronounced "sharp P complete" or "number P complete" is a complexity class in computational complexity theory. A problem is #P-complete if and only if it is in #P, and every problem in #P can be reduced to it by a polynomial-time counting reduction, i.e. a polynomial-time Turing reduction relating the cardinalities of solution sets.
more from Wikipedia
Approximation algorithm
In computer science and operations research, approximation algorithms are algorithms used to find approximate solutions to optimization problems. Approximation algorithms are often associated with NP-hard problems; since it is unlikely that there can ever be efficient polynomial time exact algorithms solving NP-hard problems, one settles for polynomial time sub-optimal solutions.
more from Wikipedia
Hamming distance
In information theory, the Hamming distance between two strings of equal length is the number of positions at which the corresponding symbols are different. Put another way, it measures the minimum number of substitutions required to change one string into the other, or the number of errors that transformed one string into the other.
more from Wikipedia
P (complexity)
In computational complexity theory, P, also known as PTIME or DTIME(n), is one of the most fundamental complexity classes. It contains all decision problems which can be solved by a deterministic Turing machine using a polynomial amount of computation time, or polynomial time.
more from Wikipedia
Cryptographic protocol
A security protocol (cryptographic protocol or encryption protocol) is an abstract or concrete protocol that performs a security-related function and applies cryptographic methods. A protocol describes how the algorithms should be used. A sufficiently detailed protocol includes details about data structures and representations, at which point it can be used to implement multiple, interoperable versions of a program.
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text: Andy: I just started reading your book "The Grand Jihad – How Islam and the left Sabotage America". Its spot on. Thank you for being a brave warrior in this battle. You remind me a lot of Patrick Buchanan! Have you considered running for political office? We need people like you running things instead of on the sidelines.


text:
Mytilus' Summer Sale
May 14, 2012 12:30 am
Tags: 2012, MarketPlace Promotions, May, Mytilus, Sale, Summer
Mytilus' Summer Sale
Open all the curtains and install the screen doors and begin to enjoy the warm evening breezes that this season has to offer! Renderosity vendor Mytilus will be spreading some sunshine around the community by offering a great opportunity to save big in their store!
From now through Friday May 18th, Mytilus has cut prices on over 60 products by 50% to 60%.
Be sure to visit Mytilus' Store during this week long event ONLY at Renderosity!
May 14th - May 18th
Save 50 - 60% throughout their entire store!
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text:
Top Definition
1. An elevated surface/stage/ledge that can be used for doing tricks on a BMX or a skateboard, particularly a manual (called a 'manny' in this context). Often times found in parking lots, they can be as low as a curb, but must be a least a few feet wide and long. Usually only riden when there is nothing else around, though serious tricks might go down on the taller ones.
Bro, there's nothing to skate around here except a little manny pad.
by T34418L31 May 16, 2006
Trending Now


text:
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Shrine Cabinet1 comment
This cabinet is 63" long and 32" high. It was designed for a client using a design line from a major shrine cabinet in a meditation center. The line was of the major shrine was designed by the...view details
The essential toolbox4 comments
This toolbox was based on the essential toolbox plans available from Fine Woodworking. I customized it by increasing the size and using different woods. The woods used are quilted maple and cocobolo.view details
Nautilus Maple Veneer Lamp
Hi Folks, This is a lamp whose design is based on the Sea Shell , the Nautilus. It was great to design , love the sea side aspect too. Hope you like.view details
Nara Desk1 comment
The Nara desk is made from figured walnut and cherry with maple drawers. Breadboard ends on the table top are draw bored and keep the piece stable. Through tenons add accents to the face of the...view details
Asian Inspired Writing Desk1 comment
The desk is made from cutoffs and includes Maple, Ash and Cherry. Black lacquer hide the mismatched wood and quartersawn zebrawood veneer add a nice accent to the piece. It was buillt largely as...view details
Two No. 4 Shaker rockers2 comments
Rockers made of tiger maple. Wood comes from Kevin Koski at www.curlymaplewood.com, a great source. It's harvested by the Amish in eastern Ohio. Kevin dries and mills it. Here dyed with waterbased...view details
Side Table of Curly Cherry and Tiger Maple
This curly cherry and tiger maple table was a wedding present for my son and his bride. It was specifically designed as a serving table with storage for wine bottles and glasses. The table's sides...view details
Charging Station Box2 comments
My charging station box eliminates the clutter from all the charging cables when charging mobile devices. A 6 outlet plug strip is hidden behind a door in the back of the box. There are two...view details
Spalted Maple Table
I purchased a log of spalted maple and made this table: spalted maple top and walnut base, 48 inches long, 15.75 inches wide, 30 inches high. Top thickness: 1.9 inches The 'elevated' design of...view details
"Imagine" Music Box
The Music Box features the song "Imagine" by John Lennon. The 18 note movement is from TheMusicHouse.com. It is a Swiss made movement by Reuge. The basic design for the box was chosen by my...view details
Wooden Balls1 comment
I was intrigued by the tip in issue #253 about making wooden balls with a belt sander and put together a box for my sander. I have a supply of ambrosia maple end cuts that I had been saving for...view details
Communion Table
This table was done as a donation to my church. The design is my own and it was constructed from Cherry with a curly maple top panel and walnut inlay. The lettering was done by a friend with a...view details
Flute Box1 comment
Made this box for my Mothers Flute. Its made from Walnut and maple. Hand made Dovetails. Just a little something I decided to do..view details
Trestle Table1 comment
I designed the "Trestle Table" to take advantage of the wonderful grain in this figured maple. I kept the design simple but elegant to highlight the wood's beauty. The edges of the top are tapered...view details
"Dad's Wood Occasional Table"
This occasional table (42"W x 12"D x30"H) is a modification of one of my previous designs. The top and shelf are of Douglas Fir saved for 75 years from a house my client's dad built in Seattle, Wa...view details
Hufschmid H6 - Highly figured flame maple top!1 comment
A new guitar I just completed for a customer. This unique creation features a very gorgeous Highly Figured flame maple top which I've stained with my unique 'Helldunkel stain', West African Ebony...view details
Garage Bench
We needed a bench in the garage for taking off shoes and boots. This garage bench was designed to fit the space adjacent to the house door. The curved end allows the bench to be right outside the...view details
On Display Too
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Edited by James Huston
Single-chain variable fragment (scFvs) antibodies are small polypeptides (~26 kD) containing the heavy (VH) and light (VL) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of VH and VL domains, further validating the here-reported expression system.
A single-chain variable fragment (scFv) is a portion of a monoclonal antibody (MW ~26 kD), in which the variable immunoglobulin domains of the heavy (VH) and light (VL) chains are connected with a flexible linker into a single polypeptide chain (Bird et al., 1988; Huston et al., 1988). Thus, an scFv contains the entire antigen-binding region, and hence the specificity, of the parent antibody (Bird et al., 1988; Huston et al., 1988; Sandhu, 1992; Holliger and Hudson, 2005). The two variable domains are tethered together in either order (VH–VL or VL–VH), with a linker that is usually 10–25 amino acids in length, spanning the 35–40 Å distance from the C-terminus of one V domain to the N-terminus of the other (Filpula et al., 1996; Weisser and Hall, 2009). The length of this linker plays a crucial role for the oligomeric state of the soluble purified scFv (Arndt et al., 1998; Filpula et al., 1996), the most common linker being a 15mer (Gly4Ser)3 (Huston et al., 1988; Weisser and Hall, 2009).
Importantly, scFvs usually bind their cognate antigens with affinity similar to that of the parent antibody (Bird et al., 1988; Huston et al., 1988, 1996; Skerra and Plückthun, 1988; Weisser and Hall, 2009)–if the avidity effect of the bivalency of the latter is taken into account. This is due to the identical 3D arrangement of the variable domains. This feature, combined with the small size of scFvs, has made them attractive candidates for a wide range of applications, including therapeutics, medical imaging and diagnostics (Begent et al., 1996). scFvs and scFv-based antibody fragments are currently in pre-clinical and clinical trials to treat human diseases ranging from heart disease to melanoma, and also for use in medical imaging (reviewed in Holliger and Hudson, 2005). In addition, scFvs have shown promise in drug delivery systems and for the targeting of gene therapy vectors (Glasgow et al., 2009; Eisenstein, 2011).
The compact fold of scFvs, composed of two immunoglobulin domains each consisting of nine strands forming two tightly packed β-sheets stabilized by an intrachain disulfide bond, provides additional protein surfaces that can help to form a crystal lattice and thus promote crystallization of macromolecules (Kovari et al., 1995; Griffin and Lawson, 2011). Such an approach is particularly useful when dealing with proteins that are difficult to crystallize, such as heavily glycosylated proteins, or multidomain proteins with relatively flexible interdomain connections. Moreover, the compactness of scFvs makes them ideal candidates for structural studies on antigen–antibody binding, such as the characterization of the neutralization mechanism of certain monoclonal antibodies in viral infection (Hwang et al., 2006; Sui et al., 2009). These studies would be extremely difficult to perform using full-length bivalent antibody molecules, and are sometimes hindered by the flexibility of the elbow angle between variable and constant domains in Fab (fragment antigen-binding) fragments.
Various expression systems for scFvs have been reported, including Escherichia coli, yeast, fungi, plants, insect and mammalian cells (Bird et al., 1988; Wu et al., 1993; Jost et al., 1994; Ridder et al., 1995; Brocks et al., 1997); reviewed in Verma et al. (1998) and Weisser and Hall (2009). Numerous studies have been performed to evaluate quantity and quality of scFvs produced in different expression systems. The advantages and drawbacks of the most frequently used expression systems are summarized in Table I. Importantly, in spite of numerous expression systems that have been explored for scFv expression, the expected effort required to produce large quantities of an scFv derived from a particular parent antibody strongly depends on the individual amino acid sequence (Verma et al., 1998). Given that it is important to consistently obtain large quantities of pure scFv for structural analyses, as well as for diagnostic or therapeutic applications, we decided to further explore the use of the Drosophila Schneider 2 system for expression of scFvs. This system has been previously used to produce scFvs, but the reported expression levels varied by almost two orders of magnitude (0.2–20 mg/l) (Mahiouz et al., 1998; Reavy et al., 2000). High-yield expression of full-length monoclonal antibodies and Fab-fragments in S2 cells has also been reported (Johansson et al., 2007a; Backovic et al., 2010). The Drosophila S2 expression system is relatively easy to handle and is based upon logarithmically growing healthy cell lines that have been transfected to achieve stable expression of recombinant protein. This is in contrast to the production in insect cells using a recombinant baculovirus, which makes a lytic infection and induces considerable stress to the infected cell. Baculoviral-aided expression can therefore lead to saturation of the endoplasmic reticulum quality control system of the cell, especially when overexpressing proteins with complex, disulfide-stabilized folds. In such cases, baculovirus-induced cell lysis results in dumping of protein that has not gone through the ER quality control into the medium, resulting in a mixture of folded and partially folded protein in the supernatant.
We undertook to establish an expression system for scFvs that would allow easy cloning of the variable domains of heavy and light chain and production of large amounts of correctly processed and secreted scFvs using Drosophila S2 cells. We created a panel of scFvs from both human and murine parent antibodies directed against various antigens. In this paper we discuss in particular the production of five of the recombinant proteins-scFv 3H5, derived from a murine antibody to Npro of bovine viral diarrhea virus (BVDV); scFv 8B9 and scFv 6A5, both derived from murine antibodies to glycoprotein E2 of BVDV; and scFvs 1:7 and A8, both derived from human antibodies to hepatitis C virus glycoprotein E2 (Allander et al., 2000).
The pMT-scFv-Strep vector was constructed based on the pT350 vector reported previously (Krey et al., 2010), containing a Drosophila metallothionein (MT) promoter, a BiP signal sequence and an enterokinase (EK) cleavage site followed by a double Strep tag (NH2-DDDDKAGWSHPQFEKGGGSGGGSGGGSWSHPQFEK-COOH). First we introduced NcoI and NotI sites by polymerase chain reaction (PCR) using oligonucleotides 5′-TTTTTTTTCCATGGCCCCGAGCGAGAGGCCAAC AAAGG-3′ and 5′-AAAAAAAAAGCGGCCGCAGACGATGACGATAAGGCCGGTTG-3′ replacing BglII and BstBI sites used for cloning. In a subsequent PCR step KpnI and NheI sites were inserted using oligonucleotides 5′-GGAGGAGCTAGCAAAAAAGCGGCCGCAGACGATGACG-3′ and 5′-TCCCGAGCCGCCGGTACCTTTTTTCCATGGCCCCGAGCGAG-3′. Finally, the complete linker sequence GGS(GGGGS)2GGG was inserted by PCR using oligonucleotides 5′-CCACCCGATCCTCCTCCTCCCGAGCCGCCGGTACCTTTTTTCCA-3′ and 5′-TGGTGGTAG CGGAGGAGGAGCTAGCAAAAAAGCGGCCGCAGACGATG-3′.
The respective DNA sequences of the individual parent antibodies have either been reported previously (1:7, A8; Allander et al., 2000) or were obtained from sequencing of the respective hybridoma cell line (8B9, 6A5, 3H5). The VH and VL of the individual scFvs were inserted into the pMT-scFv-Strep vector in two successive rounds of cloning. A VL gene was amplified by PCR using either cDNA of hybridoma cells (8B9, 6A5) or synthetic genes, codon optimized for Drosophila melanogaster (3H5, 1:7, A8), as template. The amplified segment was cloned into pMT-scFv-Strep vector using NheI and NotI. Insertion of the VL gene into the resulting pMT-scFv-#-VL-Strep constructs was confirmed by sequencing. The respective VH gene was subsequently amplified by PCR using either cDNA of hybridoma cells (8B9, 6A5) or synthetic genes, codon optimized for D.melanogaster (3H5, 1:7, A8), as template. The amplified VH sequence was cloned into the newly created VL-containing pMT-scFv-#-VL-Strep vector using NcoI and KpnI. The resulting pMT-scFv-#-Strep constructs were sequenced to confirm insertion of both the VL and the VH. A full list of oligonucleotides used for cloning of VH and VL genes is included as Supplementary material (Table S1).
Transfection of Drosophila S2 cells was done as described before (Johansson et al., in press). Briefly, we used Effectene (Qiagen, Hilden, Germany) according to the manufacturer's recommendations, to transfect parental S2 cells with 2 µg of the respective pMT-scFv-#-Strep plasmids. A second plasmid, encoding puromycin acetyltransferase, was cotransfected as dominant selectable marker. Stable scFv expressing cell lines were selected by addition of 8 µg/ml Puromycin (Invivogen, San Diego, USA) to the culture medium 72 h after transfection. Adaptation of the cell lines to serum free Insect Xpress media was performed stepwise as recommended by Invitrogen.
For large-scale production of scFvs the cells were cultured in spinner flasks and induced with 4 μM CdCl2 at a density of at least 7 × 106 cells/ml. After 7–10 days at 28°C cells were pelleted and the scFv was purified by affinity chromatography from the supernatant using a StrepTactin Superflow column (IBA, Goettingen, Germany) followed by size exclusion chromatography (SEC) using a Superdex200 column (GE Healthcare, Uppsala, Sweden). Pure protein was quantified using adsorption at UV280 nm and concentrated to ~10 mg/ml.
Dimeric scFvs were diluted to ≤100 μg/ml and dissociated by overnight dialysis in dialysis buffer (0.3 M NaCl, 20 mM Tris pH 8.0) containing 8 M urea. Subsequently, the dimeric fragments were refolded by a stepwise dialysis overnight at 4°C against 1 l dialysis buffer containing decreasing concentrations of urea (8, 6, 4, 2, 1, 0.5 and 0 M). The buffers containing 1 and 0.5 M urea were supplemented with 0.4 M l-arginine. The oligomerization state of the refolded scFvs was analyzed by SEC using a Superdex™ 200 column.
Twenty micrograms of scFv (final concentration 0.2 mg/ml) and 20–35 μg (final concentration 0.2–0.35 mg/ml) of antigen (depending on its molecular weight), were incubated as isolated proteins as well as in complex for 24 h at 4°C followed by analysis on a Superdex™ 200 5/150GL column (column volume 3 ml, GE Healthcare).
Crystals of scFv 1:7 were grown at 293 K using the sitting-drop vapor-diffusion method. Crystallization was performed using a Mosquito robot (TTP LabTech Ltd, Royston, UK) in a drop containing 800 nl protein (7 mg/ml in 10 mM TRIS pH 8.0, 150 mM NaCl) mixed with 800 nl reservoir solution containing 100 mM TRIS pH 8.5, 1550 mM (NH4)2SO4 and 200 mM Li2SO4. Diffraction quality crystals appeared after 1 week and were flash frozen in mother liquor containing 25% (v/v) glycerol. Diffraction data were collected at the Proxima1 beam line of Synchrotron Soleil and processed with XDS (Kabsch, 2010). Scaling and reduction of the data were performed using Pointless (Evans, 2005) and programs from the CCP4 suite (Collaborative Computational Project, 1994). The structure was determined by molecular replacement using Phaser (McCoy et al., 2007), an scFv derived from a human antibody served as model (PDB 3FKU; Sui et al., 2009). Model building was performed using Coot (Emsley et al., 2010) and refinement was done using AutoBuster (Bricogne et al., 2010). Figure 4 was prepared with Pymol (http://www.pymol.org). The atomic coordinates of the structural model and the corresponding structure factors were deposited in the Protein Data Bank with ID 3U6R.
The pMT-scFv-Strep vector was designed based upon an expression plasmid previously described (Krey et al., 2010) that contained an inducible Drosophila MT promoter, followed by a Drosophila BiP signal sequence, which leads to efficient translocation of the protein into the ER. At the C-terminus, this plasmid contains a double Strep-tag for efficient affinity purification and an EK cleavage site upstream of the double Strep-tag to allow for its specific removal for therapeutic applications or structural studies.
The pMT-scFv-Strep was designed to contain the VH and VL of a parent antibody in this order joined by a linker sequence (Fig. 1) that is meant to span the 35–40 Å between the C-terminus of VH and the N-terminus of VL. The major requirement for this linker is sufficient flexibility, a rationale that is followed in the most commonly used linker sequences (Huston et al., 1991). Linker sequences are typically 10–25 amino acids, shorter linker sequences have been shown to favor the formation of dimers or higher multimers (Huston et al., 1988; Arndt et al., 1998; Filpula et al., 1996; Weisser and Hall, 2009). To reduce the probability of oligomer formation, a 16-residue GGGGS-linker was designed flanked by two restriction sites coding for residues that provide a high degree of flexibility, adding up to a final linker length of 20 residues. We designed the vector to be compatible (5′ and 3′ restriction sites NcoI and NotI) to the pHEN2 plasmid frequently used for phage display selection of high-affinity antibodies (Griffiths et al., 1994) allowing for efficient transfer of selected antibodies into the pMT-scFv-Strep vector. In parallel, the linker sequence is framed by cloning sites for directional cloning of the VH and VL facilitating easy cloning of scFv fragments from hybridoma cDNA. The design of this vector permitted efficient and rapid two-step cloning of all VH and VL scFv genes as described in the Materials and methods section, generating the expression plasmids pMT-scFv-3H5-Strep, pMT-scFv-6A5-Strep, pMT-scFv-8B9-Strep, pMT-scFv-1:7-Strep and pMT-scFv-A8-Strep, respectively.
In order to generate stable S2 cell lines expressing the recombinant scFvs, S2 cells were transfected with the respective pMT-scFv-#-Strep plasmid together with a dominant-selectable marker (an expression plasmid encoding Puromycin-Acetyltransferase; Iwaki et al., 2003). After selection, cells were adapted to serum-free Insect Xpress medium (Lonza, Basel, Switzerland), amplified, and protein production was induced by the addition of 4 μM CdCl2 as described in the Materials and methods section. We purified the secreted scFvs to homogeneity from the supernatant by affinity chromatography, with yields varying between 5 and 12 mg/l supernatant. Subsequently, we subjected the eluate to SEC separating monomeric and dimeric scFv species (Fig. 2A–E). All five scFvs from human and mouse origin expressed a majority of monomeric scFv, as judged by SEC followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis under non-reducing conditions and Coomassie blue staining (Fig. 2F). In a typical SEC profile, the majority of the protein elutes at a volume corresponding to a monomer. However, depending on the individual sequence, minor peaks corresponding to dimeric and higher oligomeric scFvs could be observed (Fig. 2A–D).
The linker between the two variable domains allows the two variable regions to form the authentic heterodimer; however, it also opens up the possibility that the VH from one scFv molecule may associate with the VL from another molecule, resulting in a linear, antiparallel dimeric molecule or even forming higher oligomers (Holliger et al., 1993). Dimeric scFvs, also known as diabodies, have therapeutic applications of their own. The extreme flexibility in the Fv angle of these oligomeric forms (Holliger and Hudson, 2005) is, however, undesirable for structural studies.
Several studies have investigated the use of different refolding procedures to scFvs (reviewed in Sinacola and Robinson, 2002). To show that the dimeric protein produced in S2 cells represents diabodies rather than aggregated or misfolded protein, we pooled the dimer-containing fractions of those scFvs exhibiting an elution pattern suggestive of dimeric protein and applied a stepwise dialysis refolding approach adapted from (Sinacola and Robinson, 2002). Dimeric scFv at a concentration of 20–50 μg/ml was dialyzed against 8 M urea in the absence of reducing agents to dissociate oligomers. The urea concentration was then slowly lowered to 0 to favor the formation of monomers over dimers or multimers; l-arginine was added at the end of the process, at the lowest urea concentrations, to promote refolding and discourage the formation of aggregates (Chen et al., 2009).
SEC analysis of the refolded scFv revealed that dimeric scFvs 8B9 and 1:7 were entirely refolded into monomers (Fig. 2G and H) in the absence of any reducing agents. This indicates that the original dimeric species already contained the correct disulfide connectivity in both heavy and light chains, likely representing diabodies.
The parent antibodies of scFvs 8B9, 6A5 and A8 each bind their cognate antigen BVDV E2 and HCV E2, respectively, with high affinity as suggested by the stability of all three antibody–antigen complexes in SEC (data not shown). To demonstrate that the affinity of the monomeric scFvs to their cognate antigen was similar to that of the full-length antibody we used SEC to assess the ability of the scFvs to interact with its cognate antigen. Each scFv and its respective cognate antigen were mixed and subsequently analyzed by SEC. All three complexes of scFvs with their cognate antigen were eluted considerably earlier than the respective individual proteins (Fig. 3), indicating a stable complex formation, as observed for the parent antibodies. This was also observed for the scFv 8B9 after being subjected to the stepwise dialysis refolding procedure (data not shown), indicating that the monomeric scFv resulting from the refolding procedure also displays an antigen-binding capacity similar to the parent antibody.
One of the potential applications of a recombinant scFv is the use in structural studies to facilitate crystallization of interesting macromolecules that would not crystallize otherwise. One example of such a molecule is the major glycoprotein E2 of the Hepatitis C virus (HCV), the crystal structure of which has still not been reported despite being the focus of intense research efforts worldwide. One possible strategy to promote crystallization of this multidomain, heavily glycosylated envelope protein is cocrystallization in complex with scFvs. To demonstrate the suitability of scFvs produced in the reported S2 cell expression system for structural studies, we determined the crystal structure of the scFv 1:7, which is derived from a broadly neutralizing anti-HCV E2 antibody previously described (Allander et al., 2000; Johansson et al., 2007b). Diffraction quality crystals of the unliganded scFv, belonging to space group C2221, could be grown within 1 week. These crystals diffracted to 2.7 Å on a synchrotron source. We determined the crystal structure of the scFv using the molecular replacement method; details and statistics of the data collection, processing and refinement are given in Table II.
The scFv 1:7 displays the standard compact fold of two heterodimerizing immunoglobulin domains, VH and VL, each containing nine β-strands tightly packed in two β-sheets (Fig. 4A). The dimer interface between VH and VL buries ~558 Å2 of solvent accessible surface, which is similar to the buried surface area computed for the variable region interface of other scFvs (e.g. PDB 1qok, 2ghw) or Fab molecules (e.g. PDB 2xqy, 3n9g) of human and murine origin. A structural comparison to the database using the DALI-server (Holm and Rosenström, 2010) followed by superposition with the four closest structural neighbors (data not shown) revealed no distortion between VH and VL. Taken together, this set of evidence strongly suggests a 3D arrangement of the scFv 1:7 that is identical to the variable region of the parent antibody and thus further validates the expression system presented in this study.
Antibody diversity is generated by the combinatorial association of V, D and J segments, which in addition becomes further diversified at the actual junctions (VL–JL, VH–D and D–JH) due to imprecise joining and addition of 'N region' nucleotides. Somatic mutation, possibly driven by antigenic selection, contributes further to antibody diversity and leads to increased affinity and specificity as antibody maturation occurs (Tonegawa, 1983; French et al., 1989). The closest homologous germline genes for the 1:7 antibody suggested by immunogenetics (IMGT) V-QUEST and junction analysis (Lefranc et al., 2009) are IGHV1-69*01 F, IGHJ4*02 F and IGHD2-2*01 F in the heavy chain and IGKV3-11*01 F and IGKJ4*01 F in the light chain. The same heavy chain V gene (IGHV1-69*01 F) was found in an exceptional high frequency in anti-HCV-E2 antibodies (Allander et al., 2000; Chan et al., 2001), suggesting a preferential usage of the same V gene in the specific immune response to the HCV E2 glycoprotein. Antibodies derived from IGHV1-69*01 F have been reported to undergo somatic mutations during antigenic selection, with high frequency located in or close to the complementarity determining region (CDR) regions, indicating a positive selection for antigen binding (Chan et al., 2001). We aligned the 1:7 VH and VL amino acid sequence to the respective germline sequences (Fig. 4B) and identified somatic mutations acquired during antibody maturation. Subsequently, we mapped those mutations on the surface of the scFv structure (Fig. 4C). Mutations at the very N-terminus were disregarded, since they are very often due to the primer sequences that have been used to isolate the antibody cDNA. We found 3 mutations in the light chain and 20 mutations in the heavy chain, the majority of which clusters in or around the H1 and H2 loop, while only 2 somatic mutations are found in the long H3 loop. In the light chain two mutations are located on the L1 loop and one in the L3 loop. These data suggest a binding mode of this broadly neutralizing antibody to its cognate antigen that is predominantly mediated by H1, H2 and H3 loops and supported by the L1 loop.
The fact that the crystal structure of the scFv 1:7 could be obtained relatively easily, demonstrates that the scFvs produced in our expression system are applicable for the use in structural studies. We expressed five different scFvs derived from human and murine origin, including codon optimized as well as hybridoma-derived DNA sequences and for all five we obtained expression levels between 5 and 12 mg/l supernatant. This suggests that the strong sequence dependence that has been described for other expression systems like E.coli and mammalian cells (Jost et al., 1994; Verma et al., 1998; Weisser and Hall, 2009) is not observed in the S2 cell expression system described here. We conclude that our system is a very powerful tool, particularly for those antibodies that remain–for yet unknown reasons–difficult to obtain in large-enough yields with other expression systems.
The human antibodies A8 and 1:7 are protected in patents owned by Molecules of Man AB, a spinoff company based on discoveries made at Karolinska Institutet, Sweden. M.A.A.P. has a financial interest in the company as shareholder.
A.A.G. benefited from a Fulbright-Hays fellowship. This work was supported by the ANRS and the ANR grant ANR-2010-BLAN-1211 01 to F.A.R., in addition to the recurrent Institut Pasteur and CNRS support to F.A.R.; the Swedish Foundation for Strategic Research (Cell Factory and Infection &Vaccines programs), the Swedish Cancer Society and the Swedish Research Council to M.A.A.P. Funding to pay the Open Access publication charges for this article was provided by the Institut Pasteur.
We thank Patrick Weber of the PF6 for help with the crystallogenesis and Andrew Thompson from the beamline PROXIMA 1 at the synchrotron Soleil for help with data collection. We also thank Scott A.Jeffers for excellent advice during protein purification and Joseph J.Cockburn and Scott A.Jeffers for helpful discussions.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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